Phosphorylation of intracellular proteins is a key mechanism in the regulation of signal transduction. Kinases, enzymes that catalyze protein phosphorylation, are mediators of the signal cascades, which activate multiple pathways involving the governance of cell division and cell death. Phosphatases are enzymes that counter the activity of kinases and remove organic phosphates from their active sites on regulatory molecules, which generally cause cessation of the activation signals. The importance of protein phosphatases in cell biology is underscored by the estimation that these proteins constitute greater than 1% of all of the proteins encoded in the human genome (1). Mammalian protein phosphatases have been placed into five subfamilies, designated PP1, PP2A, PP2B, PP5 and PP7 [reviewed in (2)].
Microcystins are inhibitors of PP1 and PP2A and are generally known as hepatotoxins that result from cyanobacterial contamination of water supplies. Structurally, microcystins are cyclic heptapeptides with the basic structure cyclo (D-Ala L-X-erythro-b-methyl-D-iso-ASP-L-Y-adda-D-iso-Glu-N-methyldehydro-Ala) where L-X and L-Y represent variable L-amino acids, and Adda is the b-amino acid 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (3). The most commonly studied microcystin is microcystin LR (FIG. 6), in which the two variable amino acids are leucine and arginine. The structures of at least 50 microcystin variants have been determined (4) differing almost exclusively in the two variable residues, which can be other L-amino acids in substitution for leucine and arginine. The variable nature of these compounds suggests that they may have a spectrum of biological effects and that there are opportunities for combinatorial engineering of therapeutic microcystin compounds.
The specific hepatic toxicity of microcystins results from the restricted hepatic expression of the organic anion transporters OATP1B1, OATP1B3 and OATP1A2, which mediate the cellular uptake of microcystins. OATP1B1 and OATP1B3 transporters have previously been known as Liver Specific Transporters 1 and 2 (LST1 and LST2), respectively, in recognition of gene expression limited to the liver. The potential potency of microcystin toxins in cancer cells has been difficult to examine due to the absence of expression of these transporters in most cancer cell lines. However, there is evidence for the expression of these transporters in tumors. Western blot analyses have detected the expression of both OATP1B1 and OATP1B3 in hepatocellular carcinoma (5, 6). Also, Abe et al. (7) have reported that OATP1B1 and OATP1B3 are expressed in a few cell lines created from liver, colon, and pancreatic tumors, suggesting that there may be a wider distribution of transporter gene expression in tumors than in normal tissues.